Plasma Activation of Biological Materials for Surface Modification

ABSTRACT

Disclosed in certain embodiments is a composition comprising a biological material activated by oxygen plasma and a ligand bound to a surface of the biological material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.61/479,627 filed Apr. 27, 2011, the disclosure of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The presently disclosed subject matter includes compositions comprisingbiological material with pharmacologically active agent(s) bound to asurface thereof, either directly or through intermediary layers, andmethods for producing said compositions.

BACKGROUND

It is desirable to alter the surface chemistry of tissue and otherbiological materials in order to control the body's interaction with itafter implantation or grafting. One reason is to prevent infection.Another is to reduce inflammatory response. Yet another is to preventthe rejection of implanted biological material.

In some cases, alteration of surface chemistry can be achieved bysoaking the material in a compatible solution of a pharmacologicalagent. A number of issues arise from this approach. Once implanted, thepharmacological solution will diffuse from the material into thepatient. Generally speaking, a higher concentration that is necessaryfor local efficacy must be used due to this diffusion effect. Thesurgeon must therefore balance the total dosage of pharmacological agentwith the necessary amount required to have the desired local effect. Insome cases, concentration level required may cause undesirable sideeffects in the patient.

In addition, once implanted, it is impossible to control the rapidelution rate of the bioactive agent form the implanted tissue into theimplant site and from there, into the patient. It is generally desirablefor the bioactive agent to remain within the implanted or grafted tissuefor a certain amount of time. Further, depending on the pharmacologicalagent, the amount required in solution for local efficacy may make theimplantation or grafting procedure prohibitively expensive.

Accordingly, there remains a need in the art for biological materialssuitable for implantation or grafting into living mammals, and methodsof creating the same.

SUMMARY OF THE INVENTION

It is an object of certain embodiments of the present invention toprovide biological materials with surface modifications to allow forconjugation with a ligand.

It is an object of certain embodiments of the present invention toprovide biological materials conjugated with an antimicrobial agent.

It is an object of certain embodiments of the present invention toprovide biological materials that have reduced incidence of infectionupon implantation or grafting into a patient.

It is an object of certain embodiments of the present invention toprovide biological materials that have reduced incidence of rejectionupon implantation or grafting into a patient.

It is an object of certain embodiments of the present invention toprovide biological materials that have improved stability and integrityupon implantation or grafting into a patient.

It is an object of certain embodiments of the present invention toprovide methods of manufacturing modified biological materials asdisclosed herein.

It is an object of certain embodiments of the present invention toprovide methods of dehydrating biological materials.

The above objects of the invention, and others, may be achieved by thepresent invention which in certain embodiments is directed to acomposition comprising a biological material and a ligand bound to asurface of the biological material. The biological material can be,e.g., collagen, tissue, or bone. In certain embodiments, the tissue isacellular dermal tissue.

In certain embodiments, the present invention is directed to acomposition comprising a biological material activated by oxygen plasma.Optionally, a ligand is bound to the activated biological material.

In certain embodiments, the present invention is directed to acomposition comprising acellular tissue; a coupling agent bound to theacellular tissue; and a pharmacological agent bound to the couplingagent.

In certain embodiments, the present invention is directed to a method oftreating biological material comprising: contacting biological materialwith oxygen plasma to form activated biological material. In otherembodiments, a ligand can be bound to the activated biological material.

Where bonding is referenced herein, such bonding can be achieved throughany type of chemical bond, including, without limitation, covalentbonding, polar covalent bonding, ionic bonding, hydrogen bonding, vander Waals forces and a combination thereof.

In certain embodiments, the present invention is directed to a processfor dehydrating a biological material (e.g., an acellular biologicalmaterial).

In certain embodiments, the present invention is directed to theimplantation or grafting of a modified biological material as disclosedherein in a patient in need thereof.

In certain embodiments, the present invention is directed to a method ofperforming reconstructive surgery in a patient in need thereofcomprising implanting or grafting a modified biological material asdisclosed herein.

In certain embodiments, the present invention is directed to a method ofadministering a drug to a patient in need thereof comprising implantingor grafting a biological material conjugated with pharmacological agentor bioactive agent as disclosed herein.

In describing the present invention, the following terms are to be usedas indicated below. As used herein, the singular forms “a,” “an,” and“the” include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a pharmacological agent”includes a single pharmacological agent as well as a mixture of two ormore different pharmacological agents.

As used herein, “biological material” means any material derived inwhole or in part from an organism, including, without limitation, softtissue sources such connective and non-connective tissue. Examples ofconnective tissue includes, without limitation, tendons, ligaments,fascia, dermal tissue, fat, dura, pericardia, fibrous tissues andsynovial membranes. Examples of non-connective tissue includes, withoutlimitation, muscles, blood vessels and nerves. “Biological material”also includes hard tissue sources such as bone and cartilage. In certainembodiments, such materials may have been harvested from a livingorganism and then submitted to further processing and/or chemicaltreatment. The living organism could be comprised of eukaryotic orprokaryotic cells. Recombinant proteins, which can be derived frombacteria such as E. coli and are produced from recombinant DNA, can alsobe modified with the present invention.

“Acellular biological material” refers to biological material from whichall, or substantially all, viable cells and detectable subcellularcomponents and/or debris from cell death have been removed.

In some embodiments, the acellular biological material utilized in thepresent invention has a concentration of viable cells that is less thatabout 5%, less than about 3%, less than about 1% or less than about 0.5%of the concentration in the original biological material from which theacellular biological material was derived. In other embodiments, theacellular biological material has an amount of viable cells that is lessthat about 3%, less than about 1%, less than about 0.5% or less thanabout 0.2% of the total weight of the acellular material.

In some embodiments, the acellular biological materials utilized in thepresent invention comprise less than about 40%, less than about 25%,less than about 10%, or less than about 5% of nucleic acid that waspresent in the original cellularized biological material from which theacellular biological material was derived. In other embodiments, theacellular biological material has an amount of nucleic acid that is lessthan about 25%, less than about 10%, less than about 5%, or less thanabout 2% of the total weight of the acellular material

“Activated biological material” refers to biological material that hasbeen contacted with an activating agent (e.g., oxygen plasma) to providereactive functional groups on the surface of the material.

“Pharmacological agent” or “bioactive agent” means any agent that can bebound to activated biological material. Examples of bioactive orpharmacological agents include, without limitation, (i) those of ananti-infective nature, such as antimicrobials, antibiotics, antifungals,antiseptics, disinfectants, and preservatives; (ii) immunosuppressantdrugs such as glucocorticoids, antibodies, ciclosporin, tacrolimus,calcineurin inhibitors, and sirolimus; and (iii) agents to mediate andinduce cellular/tissue growth such as bone morphogenic proteins (BMP),epidermal growth factor (EGF), fibroblast growth factor (FGF),platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-Iand II), TGF-D, and vascular endothelial growth factor (VEGF).

“Anti-infective” refers to anything that is capable of destroying orinhibiting the microorganism growth, including, without limitation,antimicrobial agents such as antibacterial and antifungal agents.

“Coupling agent” means an agent capable of forming a bond between thesurface of an activated biological material and a ligand (such as apharmacological or bioactive agent). This bond may be achieved by firstforming a bond between the surface of the activated biological materialand the coupling agent, and then forming a bond between the couplingagent and the ligand. In other embodiments, the ligand can be bonded tothe coupling agent, and the resultant conjugate is bound to thebiological material. Alternatively, the coupling agent may facilitatebonding directly between the surface of an activated biological materialand the ligand.

The term “oxygen plasma” means an oxygen source having a portion of themolecules ionized.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic diagram of one embodiment of the presentinvention.

FIG. 2 depicts retention of gentamicin under conditions of soaking in aninfinite sink in PBS for 28 days versus untreated control. The Y-axis isnanograms per sample core. The X-axis is time.

FIG. 3 depicts the results of elution studies of (i) samples treatedaccording to the method of the present invention, (ii) untreatedsamples, (iii) samples not treated with plasma but soaked in gentamicin,and (iv) samples dehydrated and exposed to plasma and then soaked ingentamicin. FIG. 3 demonstrates that soaked samples eluted practicallyall of their gentamicin by the end of the first day and thereafter wereequivalent to untreated samples. Samples treated with plasma accordingto the present invention showed a burst elution of unbound materialfollowed by a steady state of gentamicin over the 14 days of theexperiment. Drug retention was measured by soaking the test samples inbuffer of sufficient volume to be considered an infinite sink.

FIG. 4 depicts the long-term efficacy of gentamicin in samples treatedaccording to the method of the present invention. FIG. 4 demonstratesthat explanted tissue retains antimicrobial activity even after almost 7days of implantation time

FIG. 5 depicts a graph that demonstrates gentamicin load issignificantly improved after introducing short vacuum during the CDIactivation step of the method of the present invention.

FIG. 6 depicts the antimicrobial efficacy of samples in which a shortvacuum has been introduced during the CDI activation step of the methodof the present invention versus samples prepared without vacuum.

FIG. 7 depicts bacterial outgrowth results. N=3 per group, pertimepoint. Error bars represent the standard deviation.

FIG. 8 depicts results from a fibroblast assay. N=4 per group. Errorbars represent the standard deviation.

FIG. 9 depicts cell viability results using an MTS assay. Relative cellnumber was extrapolated from a standard curve produced from dermalfibroblasts grown on TCPS. N=4 for all groups. Error bars represent thestandard deviation.

FIG. 10 depicts the results of a gentamicin quantification assay usingan ELISA kit.

FIG. 11 depicts the results of killing assays for samples treatedaccording to a method of the present invention using 4 differentbacteria. For all groups, n=3 and error bars represent the standarddeviation.

DETAILED DESCRIPTION

Harvested biological material is often utilized for implantation orgrafting in a host organism for a variety of reasons such asreconstructive surgery (e.g., hernia repair or external burn treatment).A common source of biological material is dermal tissue. The source ofthe dermal tissue can be from another area of the patient's body, calledan autograft, obtained from another person (e.g., donor skin fromcadavers called an allograft, or from an animal (e.g., porcine or bovinesource), called a xenograft.

Implanted or grafted biological material is susceptible to complications(e.g., infection) which can lead to failure of the procedure and issuesto the patient. Accordingly, it is desirable to have the biologicalmaterial functionally modified (e.g., conjugated or bound to ananti-infective agent) prior to implantation or grafting.

In certain embodiments, the biological material is animal tissue. Animaltissue is mainly made of Type I collagen, which contains a very limitednumber of functional groups for bioconjugation (approximately 5%). Ininstances where it is desirable for these tissues to be processed,altered or derivitized for implantation or grafting into an organism,this lack of functional groups can present challenges. For example, itmay be desirable to treat implanted tissues with antibiotic agents priorto implantation to prevent infection at the surgery site. However, thelack of functional groups to which these agents can bind makes itdifficult for the tissue to retain the agents long enough for the agentsto impart the desired effect—i.e., preventing infection.

The present invention provides a solution to this problem by creatingfunctional groups on the surface of biological material after exposureto an activating agent. Preferably, the activating agent is oxygenplasma. By virtue of the present invention, biological material (e.g.,tissue) with surface functional groups such as hydroxyl and carboxyl arecreated after contact with oxygen plasma. This creation of functionalgroups creates a surface on the biological material for the binding ofligands such as antimicrobial and antibiotic agents. Theplasma-generated surface reactive hydroxyl and carboxyl groups may beformed by breaking hydrocarbon bonds on the surface of the biologicalmaterial through bombardment of oxygen plasma. Preferably, the treatmentdoes not disrupt the integrity of the bulk tissue underneath, presentingminimal challenges to post-implant integration.

One method of the present invention involves contacting a biologicalmaterial with oxygen plasma to, for example, remove the water shell thatis tightly associated with the biological material and activate theexposed surface to generate more functional groups for chemicalmodification. Ligands may then be bound to these functional groups.Ligands may include coupling agents and pharmacological or bioactiveagents. In some embodiments, a coupling agent is used to facilitatebonding (e.g., covalent bonding) between the activated biologicalmaterial and a pharmacological or bioactive agent (such as anantibiotic). For example, plasma-generated surface reactive groups arebound to a coupling agent. Ligands are then directly conjugated onto thecoupling agent biological material conjugate. In a certain embodiment, acoupling agent such as 1,1′-carbonyldiimidazole can be used to create abond between the activated surface groups and free amine groups and theantibiotic gentamicin. Another coupling agent is a mixture of1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) andN-Hydroxysuccinimide (NHS). In certain embodiments, the biologicalmaterial is dehydrated prior to activation and/or ligand binding.

Intermediary layers (e.g., coupling agents) that may be used to create aseries of bonds between the plasma-activated tissue surface and thefurther ligand preferably have one or more of the following qualities:(i) in its final processed state it is biocompatible; (ii) it isreactive with the activated tissue substrate upon application; (iii)after application, the intermediary layer is reactive with the bioactiveagent or subsequent intermediary layer; and (iv) in its final processedstate, it is substantially as flexible as its underlying substrate,which may be achieved, for example, through the thinness of the layer.

Certain embodiments of the present invention include a compositioncomprising a biological material and a ligand bound (e.g., via covalentbonding) to a surface of the biological material. The biologicalmaterial may be, for example, soft tissue, collagen, bone, dermaltissue, or any other suitable tissue. In certain embodiments, thebiological material is acellular. The ligand can, for example, be apharmacological agent, a biologically active molecule, or a couplingagent.

In certain embodiments, the present invention includes a compositioncomprising acellular tissue, a coupling agent bound to the acellulartissue, and pharmacological agent bound to the coupling agent.

The biological material can be derived from any suitable biologicalsource, including, without limitation, mammalian, avian, reptilian,amphibian and bacteria. In certain embodiments, the mammalian source isselected from humans, primates (e.g., monkeys, chimpanzees, gorillas,gibbons and orangutan), livestock (e.g., pigs, cows, horses, goats,sheep), dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, andmice. In certain embodiments, the mammalian source is a human cadaver.In other embodiments, the mammalian source is a living human. Examplesof avian sources include chicken, turkey, duck and goose.

The biological material is preferably acellular as to avoid therejection of the biological material from the host. One source ofacellular tissue that can be modified according to the present inventionis FlexHD® commercially available from Ethicon. Further examples ofacellular biological materials are described in U.S. Patent ApplicationPublication No. 2006/0275377; International Application No.PCT/US08/52882; International Application No. PCT/US08/52884, andInternational Application No. PCT/US08/52885, which are incorporatedherein by reference in their entireties.

In certain embodiments, the present invention is directed to dehydratedbiological materials and methods of preparing the same. In certainembodiments, the dehydrated biological material can optionally beactivated and/or ligand bound as disclosed herein. The dehydrationprocess can include, for example, lyophilization or a solvent exchangeprocess. In some embodiments, the biological material has beendehydrated via a solvent exchange process comprising soaking thebiological material in a solvent. In further embodiments, the biologicalmaterial has been dehydrated via a solvent exchange process comprisingsoaking the biological material in a hydrophilic solvent followed bysoaking the biological material in an organic solvent. In someembodiments, the biological material has been dehydrated via a solventexchange process comprising soaking the biological material in ahydrophilic solvent followed by soaking the biological material in anorganic solvent under vacuum.

In a particular embodiment, the solvent exchange process comprisessoaking the biological material in a solvent miscible with or capable offorming an azeotrope with water. The biological material can then beoptionally placed under a vacuum with or without heat.

In another embodiment, the solvent exchange process comprises soakingthe biological material in a solvent miscible with or capable of formingan azeotrope with water followed by soaking the biological material in avolatile organic solvent. Preferably, the boiling point of the organicsolvent at atmospheric pressure is less than or equal to about 80° C.,less than or equal to about 70° C., or less than or equal to about 80°C. The biological material can then be optionally placed under a vacuumwith or without heat.

An exemplary solvent exchange process comprises soaking the biologicalmaterial in an organic hydrophilic solvent miscible with water, such asethanol, iso-propanol, or any other azeotrope-forming solvent to extractthe water from the biological material. This can be performed for one ormore cycles (e.g., 2 or 3 cycles). The tissue can then be soaked inorganic solvents with lower boiling points, such as dichloromethane ortetrahydrofuran, for one or more cycles (e.g., 2 or 3 cycles) to replacethe previous solvent. After draining out the solvent, the biologicalmaterial may be dried. In one embodiment, the sample is placed undervacuum with or without gentle heating, to remove any residual solventthat remains.

The solvent for a solvent exchange process used to prepare thebiological material of the present invention can be, for example, one ormore of ethanol, n-propanol, iso-propanol, n-butanol, sec-butanol,iso-butanol, tert-butanol, allyl alcohol, benzyl alcohol, furfurylalcohol, cyclohexanol, benzyl alcohol, tetrahydrofuran, chloroform,methyl ethyl ketone, benzene, ethyl acetate, cyclohexane, benzene,carbon tetrachloride, ethylene chloride, acetonitrile, toluene,n-hexane, n-heptane, carbon disulfide, diethyl ketone, n-propyl acetate,methanol, acetone, aqueous mixtures thereof, and combinations thereof.In certain embodiments, the solvent is an organic solvent and can be,for example, one or more of dichloromethane, tetrahydrofuran ethylether, methyl t-butyl ether, pentane, hexane, aqueous mixtures thereof,and combinations thereof.

In embodiments that subject the biological material to a solventexchange process, the biological material may be placed under vacuum atroom temperature or with heat.

In certain embodiments of the present invention, the ligand that isbound to the biological material is a coupling agent. The coupling agentcan, for example, be 1,1′-carbonyldiimidazole,1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride,N,N′-Disuccinimidyl carbonate, N-hydroxysuccimimidyl chloroformate,isocyanate, Benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate, (Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate,O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, O-(Benzotriazol-1-yl)-N,N,N,′N′-tetramethyluroniumhexafluorophosphate, O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate, (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate, 1-Hydroxybenzotriazole,N,N′-Dicyclohexylcarbodiimide, N,N′-Diisopropylcarbodiimide, Diethylazodicarboxylate or N,N′-Di-tert-butylcarbodiimide, salts thereof,derivatives thereof and combinations thereof.

In some embodiments, a second ligand is bound to the composition of thepresent invention. The second ligand can, for example, be apharmacological agent that is bound (e.g., covalently bound) to thebiological material via the first ligand. The pharmacological agent canbe, for example, an antimicrobial agent such as an antibiotic. Incertain embodiments, the antibiotic has an available nucleophilic group.

In certain embodiments, the antimicrobial agent is selected from one ormore of amikacin, gentamicin, kanamycin, neomycin, netilmicin,tobramycin, paromomycin, geldanamycin, herbimycin, loracarbef,ertapenem, doripenem, imipenem/cilastatin, meropenem, cefadroxil,cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin,cefprozil, cefuroxime, cefditoren, cefoperazone, cefotaxime,cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone,cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin,telavancin, clindamycin, lincomycin, daptomycin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,troleandomycin, telithromycin, spectinomycin, spiramycin, aztreonam,furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,methicillin, nafcillin, oxacillin, penicillin G, penicillin V,piperacillin, temocillin, ticarcillin, amoxicillin/clavulanate,ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate,bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid,norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin,temafloxacin, mafenide, sulfonamidochrysoidine, sulfacetamide,sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole,sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline,oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin,cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide,rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine,chloramphenicol, fosfomycin, fusidic acid, linezolid, metronidazole,mupirocin, platensimycin, quinupristin/dalfopristin, rifaximin,thiamphenicol, tigecycline, tinidazole, salts thereof, derivativesthereof, and combinations thereof.

In certain embodiments, the antimicrobial agent of the present inventionis selected from one or more of chlorhexidine, biguanides, quaternaryammonium compounds, salts thereof, derivatives thereof, and combinationsthereof.

The attachment of the ligand can be performed, e.g., by contact with anaqueous, non-aqueous or partial aqueous solution of the ligand. Thesolution can contain the ligand, e.g., in an amount from about 0.0001%to about 99% w/w. In particular embodiments, the ligand solution (e.g.,gentamycin or 1,1′-carbonyldiimidazole) comprises from about 0.0001% toabout 50%, from about 0.001 to about 25%, from about 0.01 to about 10%or from about 0.1 to about 5% ligand.

In certain embodiments, the source of the oxygen plasma can be O₂, air,or a combination thereof. In other embodiments, the source of oxygenplasma is any gaseous mixture that has a minimum percent of oxygen toprovide a suitable surface of functional groups on the biologicalmaterial after contact to allow for further chemical modification

In certain embodiments, a ligand such as an antimicrobial or antibioticagent is directly bound to the surface functional groups of thebiological material without the use of intermediary layers such ascoupling agents.

In certain embodiments, the compositions, biological materials, oractivated biological materials are sterilized, i.e., they aresubstantially free of living microorganisms such as bacteria andviruses.

In certain embodiments of the inventive composition comprising apharmacological agent, the composition maintains between about 5% and95% of the pharmacological agent after soaking in an infinite sink inphosphate buffer saline for 24 hours, for 4 days, for 7 days, or for 14days.

In further embodiments of the inventive composition comprising apharmacological agent, the composition maintains between about 50% and95% of the pharmacological agent after soaking in an infinite sink inphosphate buffer saline for 24 hours, for 4 days, for 7 days, or for 14days.

In other embodiments of the inventive composition comprising apharmacological agent, the composition maintains at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90% ofthe pharmacological agent after soaking in an infinite sink in phosphatebuffer saline for 24 hours, for 4 days, for 7 days, or for 14 days.

In further embodiments of the inventive composition comprising apharmacological agent, the amount of pharmacological agent is maintainedafter soaking in an infinite sink in phosphate buffer saline for 4 days,for 7 days, or for 14 days is within about 10%, within about 15%, withinabout 20%, or within about 25% of the amount of pharmacological agentpresent in the composition at 24 hours.

The modified biological materials disclosed herein can be used inreconstructive surgery including but not limited to hernia repair,breast reconstruction, abdominal wall repair, chest wall repair,urological repair, bone and cartilage implantation, gynecologicalrepair, plastic surgery, tendon repair, burn and wound treatment andvein/artery repair.

The modified biological materials disclosed herein are optionallypackaged in a sterile container for transport and storage.

The following examples are set forth to assist in understanding theinvention and should not be construed as specifically limiting theinvention described and claimed herein. Such variations of theinvention, including the substitution of all equivalents now known orlater developed, which would be within the purview of those skilled inthe art, and changes in formulation or minor changes in experimentaldesign, are to be considered to fall within the scope of the inventionincorporated herein.

EXAMPLES Example 1 Solvent Exchange

FlexHD® samples were immersed in absolute ethanol for 15 minutes withgentle shaking. This step was repeated until a total of 3 cycles hadbeen completed. The samples were then immersed in dichloromethane for 15minutes with gentle shaking. This step was repeated once more so that atotal of 2 cycles were completed. The samples were then placed in avacuum oven at 30° C. for 1 hour to remove residual solvent.

Example 2 Plasma Activation

Lyophilized acellular biological material, or a sample dehydratedaccording to Example 1, was placed inside a plasma chamber using air oroxygen as the process gas. When the pressure inside the chamber wasbelow 2 Torr, the plasma process was started. The samples were treatedby plasma for 0.5 to 5 minutes depending on sample size, with the radiofrequency level set to “HIGH” (30W).

Example 3 Attachment of Coupler to Activated Sample

Immediately after processing according to Example 2, samples wereimmersed in 50 mL of tetrahydrofuran (THF) containing 175 mg of1,1′-carbonyldiimidazole (CDI) for 30 minutes in a 50 mL Falcon tube atroom temperature to yield samples to which CDI was chemically bound tothe samples. For lyophilized samples, this example was carried out in adesiccator connected to a vacuum pump, vacuumed to a pressure of about30-60 Torr for about 5-15 minutes to remove air bubbles inside thesample.

Example 4 Attachment of Antibiotic

The CDI-bound samples of Example 3 were rinsed with dry THF and immersedin 50 mL of an aqueous gentamicin solution (10 mg/mL) at roomtemperature for 2 hours to bind the gentamicin to the sample. Followingthe gentamicin conjugation, the tissue samples were washed withdeionized water and then soaked in 1× PBS for at least 16 hours toremove any unbound gentamicin.

Example 5 Gentamicin Retention vs. Untreated Control

Samples prepared according to Example 4 were soaked in an infinite sinkin PBS for 28 days. Untreated controls were also soaked under the sameconditions. Gentamicin content (in nanograms per sample) was measured invitro at 0, 24, 48, 168, 336, and 672 hours (Day 0, Day 1, Day 2, Day 7,Day 14, and Day 28, respectively). FIG. 2 depicts the retention ofgentamicin in samples taken at these points in time. As shown, thegentamicin content per sample dropped drastically between Day 0 and Day1, which reflects the initial elution of unbound gentamicin from thesample. The amount of gentamicin per sample remained steadily betweenabout 1500 ng and 500 ng from Day 1 to Day 28.

Example 6 Gentamicin Retention vs. Soaked Control

Samples prepared according to Example 4 were compared with: (i) samplesthat were soaked in gentamicin but not treated with oxygen plasma orCDI, (ii) samples that were dehydrated and exposed to air plasma (butnot treated with CDI) and then soaked in gentamicin, and (iii) samplesthat were not treated at all. All samples were soaked in an infinitesink in PBS for 14 days. Gentamicin content (in nanograms per sample)was measured in vitro at 0, 24, 96, 168, and 336 hours (Day 0, Day 1,Day 4, Day 7, and Day 14, respectively). FIG. 3 depicts the retention ofgentamicin in samples taken at these points in time. As shown, thegentamicin content of the treated sample dropped drastically between Day0 and Day 1, which reflects the initial elution of unbound gentamicinfrom the sample. The amount of gentamicin in the treated sample remainedsteadily between about 70 ng and 85 ng from Day 1 to Day 14. After Day0, the control samples drastically dropped to no more than about 10 ngof gentamicin from Day 1 to Day 14. This data demonstrates thatpreparing a sample according to the direct plasma process described inExamples 1-4 enables it to retain significantly more gentamicin thanjust soaking the sample in gentamicin in the absence of CDI and oxygenplasma.

Example 7 Bacterial Outgrowth with E. Coli

Samples prepared according to Example 4, as well as untreated controls,were implanted into non-inoculated subcutaneous pockets of rats for aset amount of time and then explanted. The treated and untreated sampleswere then evaluated in an outgrowth assay in which bacteria were seededonto the samples and then placed in bacterial growth media. FIG. 4depicts bacterial growth in each of the treated and untreated samples atDay 0, Day 4, Day 7, and Day 14. As shown, the explanted treated tissueretains antimicrobial activity after almost 7 days of implantation time.

It is noted that although the in vitro data from Examples 5 and 6demonstrate that gentamicin was retained in the sample for at least 28days, the in vivo data of this Example 7 shows cessation of efficacyafter 7 days. This can be explained by the natural remodeling processgoing on in the organism's body. As soon as the sample is implanted, itbecomes coated with serum proteins as the body begins to incorporate andremodel it. Over time, the foreign material will become coated with newconnective tissue and nascent vasculature. In the case of the explantedtissue, by Day 7, the sample has already been coated with connectedtissue and is well on its way to being remodeled. In other words,although the gentamicin in the matrix of the sample is likely stillbound, it has been sequestered by the newly formed connective tissue andis not exposed to the bacterial colonies in the outgrowth assay.

Example 8 Attachment of Antibiotic Using a Vacuum

Plasma-activated samples were placed in solution containing CDI. Forlyophilized samples, the solution was placed in a desiccator connectedto a vacuum pump, and the desiccator was vacuumed to a pressure of about30 to 60 Torr for 5 to 15 minutes to remove air bubbles inside thesample to achieve better solution penetration. After 30 minutes to 2hours of activation under agitation, samples were immersed in anothersolution containing a target bioactive molecule for 2 to 8 hours underagitation in order to attach the molecule to the surface of the sample.The samples were then washed with deionized water 5 to 10 times toremove any unattached molecules.

FIG. 5 shows that gentamicin retention is significantly improved afterintroducing a short vacuum during the CDI activation step. Theantimicrobial efficacy is also enhanced dramatically in the bacteriaoutgrowth assay, as shown in FIG. 6.

Example 9 Bacterial Outgrowth Assay

An overnight culture of E. coli was diluted down to OD₆₀₀ nm=0.03(approximately 10⁶ CFU/mL) in LB (Lennox) broth (Fisher) and 20 μL ofthis bacterial dilution was pipetted onto 6 mm biopsy punches ofuntreated human dermis as a control or plasma gentamicin-treated dermis.The samples incubated for 10 minutes at room temperature before each onewas placed into a sterile 50 mL Erlenmeyer flask containing 10 mL LBmedia. The flasks were shaken at 37° C. at 225 RPM in an orbital shaker(Lab-Line), and optical density measurements were taken every hour tomonitor bacterial growth until turbidity was achieved or no growth wasobserved.

As depicted in FIG. 7, at 2, 3, and 4 hours of outgrowth assay, therewere elevated numbers of E. coli in the untreated tissue compared withthe treated samples. The control samples showed an exponential growthpattern while the treated samples showed no growth, indicative ofcomplete bacterial eradication during the soaking period.

Example 10 Fibroblast Biocompatibility

Human dermal fibroblasts derived from neonatal foreskin were grown toconfluency at 37° C. in a CO₂ incubation chamber, in essential mediasupplemented with 10% FBS and pen/strep and split in a 1:10 dilution.All studies were performed with passage four cells. Plasma treated andcontrols samples were prepared using 6 mm skin biopsy cores to createtest samples. The samples were soaked in 70% EtOH for 30 minutes andthen placed into essential media for an overnight soak for at least 16hours. The soak media was aspirated and the samples were placedindividually into wells of a 24-well plate and seeded with 10,000fibroblasts per sample delivered in 50 μL of complete media. After 30minutes, an additional 500 μL of complete media was added to each well.Cells were assayed for metabolic activity at 1 hour and 24 hours afterpost-seeding using the TACS MTT assay (Trevigen, Md., USA) according tothe manufacturer's instructions. Standard curves were seeded 24 hoursprior to performing the MTT assay for the 24 hour samples. For the 1hour samples, the standard curve was prepared at the time of testarticle preparation, which may not have allowed for metabolic activityto resume. For each time point, n=4 treated and control samples weretested. In addition four treated and control samples were measured forMTT activity without being cell seeded.

Readings from these samples were not different between treated anduntreated test samples. Therefore, the dermis background to besubtracted from the cell seeding values was determined by averagingthese 8 samples together. The MTT activity for the 1 hour samples wasdetermined by assaying the dermis and media samples together, however,it was observed that significant portion of the formazin product wassequestered in the dermis. Therefore, for the 24 hour study, the mediaand dermis were assayed separately and background form control dermiswas also assayed separately.

The results of the fibroblast assay are shown in FIG. 8. There was nosignificant different between the treated and control samples at eithertime point.

Example 11 Cell Viability

Cell viability was assessed at 1 and 4 days of exposure to the dermalmatrices. For each timepoint, eight samples from each test group wereplaced with the epidermal surface facing up into the wells of a 96 wellTCPS plate. The samples were washed with 100 μL of complete media for 5minutes followed by aspiration of the media and placement of eithercells or fresh media without cells onto the samples. For the cell-seededsamples, 10,000 human dermal fibroblasts were seeded onto 4 of the 8human dermis test samples in 100 μL of complete media.

At the time of initial test sample cell-seeding for the 1 day cellviability samples, a standard curve was prepared in duplicate using thehuman dermal fibroblasts. To produce the standard curve, 25,000, 12,500,and 6,250 cells were seeded directly onto the TCPS wells. For the 4 daystudy, the standard curve was prepared three days post cell seeding. Themedia was exchanged every 24 hours during the study and just prior tothe termination of the assay.

The CellTiter 96 Aqueous One Solution Assay (Promega, USA) was used toassess cell viability at 1 and 4 days post cell seeding. The kit wasused according to the manufacturer's instruction with a few minormodifications made to accommodate use with human dermis tissue. To eachwell of the standard curve and the test samples, 20 μL of MTS solutionwas added. After 90 minutes, the dermis was gently transferred to cleanwells leaving the media behind. The colorimetric intensity of each wellwas measured at 490 nm in both the dermis and media containing wells.The non-cell seeded blanks were averaged together for both the media anddermis and subtracted from the cell-seeded samples to obtain normalizedreadings. It was necessary to use separate blanks for each of thetreatment because it was observed that modifications made to the tissuecould change the background color levels of both the media and dermis.The total normalized color observed in the media and dermis was addedtogether and the cell number was determined using the standard curve.

FIG. 9 depicts the results using the MTS assay. Relative cell number wasextrapolated from a standard curve produced from dermal fibroblastsgrown on TCPS. There was no significant difference between control andplasma treated samples and controls samples at either point in time.

Example 12 Gentamicin Quantification

The amount of gentamicin loaded into the human dermis was measured insamples prepared as described in Example 11. Three separate dermissamples from the control and plasma groups were weighed just prior togentamicin determination. The samples were placed into 5 mL of 0.25M HClin borosilicate test tubes covered with aluminum foil. The samples wereautoclaved at 121° C. with a 1 hour cycle in a Steris autoclave (SG-120Scientific Gravity Sterilizer) to degrade and solubilize the collagen.In earlier studies, the stability of gentamicin in 0.25M HCl and underautoclave conditions was assessed. No significant loss of gentamicindetection was observed.

Following the autoclave step, the gentamicin was measured using acommercially available ELISA kit (BioO, USA) according to theinstruction included in the kit. Human dermis samples were handledaccording to the protocol as if they were milk samples. Usinganticipated drug loads determined from earlier zone of inhibitionstudies, the autoclaved samples were diluted into 1× sample extractionbuffer 1:40. Standards provided with the kit were used to create astandard curve and subsequently to calculate total gentamicin in eachsample. The gentamicin load was expressed as parts per million (ppm) bydividing the weight of gentamicin by the weight of the dermis sample.

FIG. 10 depicts the results of the assay. Plasma treated samplescontained an average of 24 ppm of residual gentamcin. The controltissue, which did not contain any gentamicin, showed an ELISA value of0.1 ppm, which represents the baseline background level inherent to theassay.

Example 13 Antimicrobial Activity

The experimental procedure described in the quantitative method of ASTM2180 was modified and used to evaluate the antimicrobial effectivenessof gentamicin bound to treated and untreated human dermis. The surfacearea of the test samples was reduced from 3×3 cm squares to 6 mmdiameter round dermal biopsy cores. The slurry inoculum volume wasreduced from 0.5-1.0 to 6 μL, which provided the required 1mm depth ofslurry across the sample. The neutralizing broth volume was thereforereduced to 600 μL. Finally, the duration of the experiment was increasedfrom 24 hours to 48 hours of exposure of the inoculum to the testsurfaces. Consequently, the moisture level of the dermis samples had tobe controlled by placing the samples on hydrated squares of sterilefilter paper moistened with 200 μL sterile PBS in order to recoverviable cells from the untreated samples at 2 days post-slurryinoculation. The filter paper squares were checked form moisture leveldaily and approximately 200 μL of PBS was added each day.

Briefly, 18 hour bacterial cultures of S. aureus (ATCC 25923 and 10390),E. coli (ATCC 25404), or P. aeruginosa (ATCC 27853) were grown anddiluted to OD₆₀₀=1.0, 0.5, 0.5, or 0.5, respectively, in an agar slurrycontaining 0.85% NaCl and 0.3% agar which was sterilized andequilibrated in a water bath to 45° C. Treated and untreated (control)dermal biopsy cores (in triplicate for each time point) were placed intosterile 35×10 mm petri dishes containing 2.4 cm² sterile filter papers,and 6 μL of inoculated slurry was pipetted onto each sample. The sampleswere allowed to gel for 10 minutes at room temperature before 200 μLsterile PBS was pipetted onto the filter paper squares to keep the coreshydrated. The petri dishes were placed into the 37° C. incubator for aspecified contact time (1 hour, 1 day, or 2 days). Following thespecified contact time, the untreated and treated samples were collectedin 600 μl neutralizing broth (TSB for S. aureus and P. aeruginosa; LBfor E. coli) to form a 1:100 dilution of the initial inoculum. Thesamples were sonicated for 1 min in a non-cavitating sonic bath,followed by 1 min of vigorous mechanical vortexing to release the agarslurry from the samples. Serial dilutions were performed with theneutralizing broth, plated (TSA for S. aureus and P. aeruginosa; LB forE. coli), and incubated overnight at 37° C. Percent reduction wascalculated by comparing the CFU recovered from the untreated versustreated samples.

K. pneumoniae, C. perfringens and P. mirabilis were tested using almostidentical conditions with a 24 hour contact time at Gibraltar Labs inFairfield, N.J. For their studies, all bacteria were cultured for 18hours and used at OD 0.5 in the agar slurry. The only other differencewas that 1 ml was used as the volume for the neutralizing broth in theGibraltar studies.

The internal ASTM 2180 experiments examined the antimicrobial effects ofplasma treated tissue at three different exposure times (1 hour, 1 day,and 2 days) against E. coli, P. aeruginosa, and S. aureus 25923 and10390. The percent killing and log bacterial reduction for the plasmatreated samples are shown below in Table 1, while the percent killingand standard deviation for the plasma treated sample data set are shownin FIG. 11.

TABLE 1 Percent killing and log bacterial reduction for plasma treatedsamples Bacterial Percent Killing Log Reduction Species 1 Hour 1 Day 2Days 1 Hour 1 Day 2 Days E. coli 25404 99.94 100 100 3.19 4.73 5.34 P.aeruginosa 100 100 100 4.75 6.13 6.64 27853 S. aureus 96 99.99 99.991.36 4.67 4.64 25923 S. aureus 81.3 99.7 99.9 0.72 2.49 2.84 10390

The external ASTM 2180 experiments performed at Gibraltar Labs inFairfield, N.J. utilized a single 24 hour (1 day) exposure time of thebacterial slurry on the tissue and three bacterial strains:Klebsiellapenumoniae, Proteus mirabilis, and Clostridium perfringens(vegetative cells). They tested the antimicrobial activity of plasmatreated dermis. The percent killing and log bacterial reduction forthese treated samples can be found in Table 2 below. Both of the treatedsamples resulted in 99.999% killing with a 5-log reduction of K.pneumonia, 99.99% killing with a 4.34-log reduction of Proteusmirabilis, and 99.999% killing with a 5.61-log reduction of C.perfringens. Both treated tissue samples were effective surfaceantimicrobial against all three of these bacterial species with a 24hour contact time.

TABLE 2 Percent killing and log bacterial reduction in plasma treateddermis against three bacterial strains through external ASTM 2180testing. Bacterial Species Percent Killing Log Reduction Klebsiellapneumoniae 99.999 5 Proteus mirabilis 99.99 4.34 Clostridium perfringens99.999 5.61 (vegetative cells)

Example 14 Bone Tissue Treatment Process

Bone samples were soaked in PBS or 0.9% saline solution at 37° C. forthree days. Bone samples were then immersed in 100% ethanol with gentleshaking for 15 minutes. This immersion step was repeated until a totalof three cycles had been completed. Finally the bone samples were vacuumdried at room temperature for 2 hours.

The dried bone samples were them treated with oxygen plasma for 30seconds. Immediately thereafter, the bones were immersed in1,1′-carbonyldiimidazole solution for 30 minutes, and then immersed in a10 mg/mL gentamicin aqueous solution for 2 hours followed by washingwith deionized water. Finally, the bone samples were soaked in PBS forthree days to remove any unbound gentamicin.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.

1. A composition comprising: a biological material activated by oxygenplasma and a ligand bound to a surface of the biological material. 2.The composition of claim 1, wherein the biological material is collagen.3. The composition of claim 1, wherein the biological material istissue.
 4. The composition of claim 1, wherein the biological materialis bone.
 5. The composition of claim 3, wherein the tissue is dermaltissue.
 6. The composition of claim 5, wherein the dermal tissue isacellular.
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. The compositionof claim 8, wherein the dermal tissue is derived from a human cadaver.11. The composition of claim 1, wherein the biological material isdehydrated.
 12. (canceled)
 13. The composition of claim 11, wherein thebiological material is dehydrated by a solvent exchange process. 14.(canceled)
 15. (canceled)
 16. The composition of claim 13, wherein thesolvent exchange process comprises soaking the biological material in ahydrophilic solvent followed by soaking the biological material in anorganic solvent followed by placing the biological material undervacuum.
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. The compositionof claim 1, wherein the ligand is a coupling agent.
 21. The compositionof claim 20, wherein the coupling agent is selected from the groupconsisting of 1,1′-carbonyldiimidazole,1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride,N,N′-disuccinimidyl carbonate, N-hydroxysuccimimidyl chloroformate,isocyanate, salts thereof, and a combination thereof.
 22. Thecomposition of claim 1, further comprising a second ligand bonded to thecomposition.
 23. The composition of claim 22, wherein the second ligandis a pharmacological agent.
 24. (canceled)
 25. (canceled)
 26. Thecomposition of claim 23, wherein the pharmacological agent is anantimicrobial agent.
 27. The composition of claim 26, wherein theantimicrobial agent is an antibiotic.
 28. The composition of claim 27,wherein the antibiotic has an available nucleophilic group.
 29. Thecomposition of claim 26, wherein the antimicrobial agent is selectedfrom the group consisting of amikacin, gentamicin, kanamycin, neomycin,netilmicin, tobramycin, paromomycin, geldanamycin, herbimycin,loracarbef, ertapenem, doripenem, imipenem/cilastatin, meropenem,cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole,cefoxitin, cefprozil, cefuroxime, cefditoren, cefoperazone, cefotaxime,cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone,cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin,telavancin, clindamycin, lincomycin, daptomycin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,troleandomycin, telithromycin, spectinomycin, spiramycin, aztreonam,furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,methicillin, nafcillin, oxacillin, penicillin G, penicillin V,piperacillin, temocillin, ticarcillin, amoxicillin/clavulanate,ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate,bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid,norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin,temafloxacin, mafenide, sulfonamidochrysoidine, sulfacetamide,sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole,sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline,oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin,cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide,rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine,chloramphenicol, fosfomycin, fusidic acid, linezolid, metronidazole,mupirocin, platensimycin, quinupristin/dalfopristin, rifaximin,thiamphenicol, tigecycline, tinidazole, pharmaceutically acceptablesalts thereof, and a combination thereof.
 30. The composition of claim26, wherein the antimicrobial agent is selected from the groupconsisting of the list of chlorhexidine, biguanides, quaternary ammoniumcompounds, pharmaceutically acceptable salts thereof, and a combinationthereof.
 31. The composition of claim 1, wherein the activatedbiological material has surface hydroxyl groups, surface carboxylgroups, or a combination thereof.
 32. The composition of claim 1,wherein the source of oxygen plasma is O₂.
 33. The composition of claim1, wherein the source of oxygen plasma is air.
 34. (canceled)
 35. Thecomposition of claim 23, wherein from about 5% to about 95% of thepharmacological agent is maintained in the composition after soaking inan infinite sink in phosphate buffer saline for 24 hours.
 36. Thecomposition of claim 23, wherein from about 50% to about 95% of thepharmacological agent is maintained in the composition after soaking inan infinite sink in phosphate buffer saline for 24 hours.
 37. (canceled)38. (canceled)
 39. (canceled)
 40. (canceled)
 41. The composition ofclaim 23, wherein from about 5% to about 95% of the pharmacologicalagent is maintained in the composition after soaking in an infinite sinkin phosphate buffer saline for 14 days hours.
 42. The composition ofclaim 23, wherein from about 50% to about 95% of the pharmacologicalagent is maintained in the composition after soaking in an infinite sinkin phosphate buffer saline for 14 hours.
 43. (canceled)
 44. (canceled)45. (canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled)
 49. Acomposition comprising a biological material activated by oxygen plasma.50. A composition comprising: acellular tissue; a coupling agent boundto the acellular tissue; and a pharmacological agent bound to thecoupling agent.
 51. A method of treating biological material comprising:contacting biological material with oxygen plasma to form activatedbiological material. 52.-98. (canceled)
 99. A method of performingreconstructive surgery in a patient in need thereof comprisingimplanting or grafting a composition of claim 1 in a patient in needthereof.
 100. A method of administering a drug to a patient in needthereof comprising implanting or grafting a composition of claim 1 in apatient in need thereof.
 101. A method of dehydrating a biologicalmaterial comprising soaking the biological material in a hydrophilicsolvent followed by soaking the biological material in an organicsolvent. 102-105. (canceled)